Field of the Invention
[0001] This invention relates to the use of a natural extract comprising polyphenols in
the manufacture of a nutritional composition for reducing the allergic symptoms of
allergic patients when exposed to allergens.
Background to the Invention
[0002] Allergies are among the most common health problem affecting the life of patients
of all age. Allergic diseases are nowadays recognized as an epidemic by the WHO. The
prevalence of allergies has been shown to increase in the past decades. Modern life
style, especially urban, has been associated with high prevalence and higher severity
of allergic manifestations.
[0003] Allergic sensitization in childhood, especially in early childhood and especially
to food allergens, is critical and of highest interest as development of an "allergic
phenotype" or "atopy" has been shown to facilitate subsequent sensitization to other
allergens. Hence allergies in childhood can be the first step of an allergic cascade
leading to multiple allergies later in life, a process commonly referred to as "The
Atopic March". For example, it has been demonstrated in human cohorts that children
with persistent food hypersensitivity early in life have a dramatically increased
risk to develop allergic rhinitis (hay fever) or asthma later in childhood (Ostblöm
et al 2008). Children with milder forms of food hypersensitivity also have increased
risk for development of respiratory allergies but to a lesser degree than children
with persistent food hypersensitivity. Therefore, attenuating the severity of food
hypersensitivity may be crucial for slowing down the "Atopic March".
[0004] In this context the management of allergic episodes and prevention of allergies are,
in childhood and infancy, of the highest importance.
[0005] The immune system of infants is actively developing all along the few first years
of life. Acting on, preventing, avoiding, managing, reducing or modulating the allergic
reactions in such young patients can influence their allergic profile short term but
also longer term for later in life.
[0006] Prevention of allergies can be achieved on different levels:
"Primary prevention" is the effect of preventing or reducing the risk of sensitization
of patients to allergens, characterized by absence or reduced levels of allergen-specific
IgE antibodies. Preventing or reducing sensitization will result in absence or reduction
of allergic symptoms upon exposure to the same allergen. By modulating the way a patient
gets sensitized in regard to one allergen or one group of allergens (primary prevention),
the subsequent allergic response may also be modulated.
"Secondary prevention" is the effect of modulating the symptoms of allergies, .i.e.
the occurrence or intensity of the allergic reaction in patient already sensitized
to one or several allergens when the patient is re-exposed to said allergen(s). By
modulating the occurrence or intensity of the allergic symptoms (secondary prevention),
the inconvenience associated with allergies is minimized.
[0007] Given these distinct concepts of allergy prevention it may be hypothesized that by
virtue of their inherent mechanisms of action, some compounds might act solely at
one or at both of these specific levels of prevention. Some may, for example, solely
reduce the sensitization to a specific allergen (primary prevention), while other
compounds may solely have merely an effect on the secondary prevention and reduce
the severity of allergic reactions. Other compounds may be able to influence both
sensitization and symptoms and thus are effective in promoting primary and secondary
prevention.
[0008] Food allergens are among the first allergens that infants encounter in their early
life: typically, cow's milk proteins may be encountered by infants not receiving exclusive
breast-feeding. Milk-proteins are indeed among the most frequently observed cause
for food allergy in infancy, followed by eggs and wheat proteins. In general, food
allergies can manifest by cutaneous (rash, eczema, others) and gastrointestinal symptoms
(abdominal cramps, pain, especially in the abdomen, vomiting) in infants and young
children. Further sensitization and episodes of allergies can also appear when the
infant/young child is exposed to a novel food such as cereals, vegetables or fruits.
[0009] Polyphenols are a large group of small compounds with anti-oxidant activity that
are commonly found in plants and fruits. Flavonoids are a subgroup of polyphenols
with shared structural features that themselves can be further sub-grouped (again
based on common structures) for examples into flavonols, flavanols, flavones, flavanones
and others. Compounds contained in apples have been used and, demonstrated to have
a general effect on allergies: flavonoids such as quercetin as well as other polyphenols
have been suspected to provide a beneficial effect on allergies in general. Apples
are rich in various polyphenols, including quercetin and phlorizidin.
In vitro and in vivo experiments in laboratory animals suggested that some polyphenols might be able to
reduce allergic reactions via a mechanism similar to common anti-histaminics (i.e.
inhibitory effect on mast-cell degranulation). This has been described in particular
in
M. Kimata et al., Clin.Exp.Allergy 2000; 30: 501-508;
M. P. Nair et al., Biochim.Biophys.Acta 2002; 1593: 29-36 and
W. Dorsch et al., Int.Arch.Allergy.Immunol. 1992; 97: 1-7. Furthermore Kojima et al describe in
Allergology International (2000) 49:69-73 that apple polyphenols can have a beneficial effect on atopic dermatitis. In
Biosci. Biotechno, Biochem. 69 69(4), 829-832, 2005, Kishi et al. describe the efficacy of apple polyphenol for treating cedar pollinosis.
[0010] WO2006003750 describes the use of cow's milk or dairy drinks containing polyphenols derived from
apple for preventing inflammation and reducing sneezing after intra-nasal administration
of ovalbumin solution, a common food allergen.
[0011] JP2009060800 discloses the use of a drink containing an extraction powder from apples for reducing
chronic urticaria induced by food allergens in male patients.
[0012] Akiyama et al. (FEBS Letters 2005) describes the use of polyphenols from apples to prevent the development of food
allergies induced by ovalbumin. The teaching of said document also refers to the administration
of polyphenols from apples in early infancy, therefore rendering the subject-matter
of the present application obvious.
[0013] JP2006096694 teaches the addition of polyphenols from apples, such as proanthocyanidins, to foods,
such as beverages or ice cream, for the prevention and treatment of allergic symptoms
induced by ovalbumin.
[0015] However there remains a need for specifically reducing allergic reaction and symptoms
especially in the population of young children and infants.
[0016] The question of food born allergic reaction in the young age is even further complicated
by the specific nutritional needs of infants and young children. Caloric intake, caloric
density, variety of desirable nutrient, protein content and protein quality are all
among the factors important to bring the most appropriate nutrition to infants and
young children. The presence of micronutrients such as vitamins and minerals are also
of importance, more specifically when their presence is limited by specific defined
ranges corresponding to the age of the target patients. As such, for the young children
and infants, the matrices of food delivery are inherently complex but they are also
of less variety: for example infants, although potentially allergic, require a specific
protein balance in a matrix of milk-derived proteins. The presence of such multiple
nutrients can potentiate the effect of the food allergens. In such complex matrices,
low key food allergens unable to trigger allergic manifestation alone, can then become
more powerful at initiating an allergic reaction. Similarly compounds that may neutralize
allergens or act on allergy prevention may see their effect diminished or annihilated
in such complex nutritional matrices. Specifically it is not predictable whether compounds
usually recognized to act on allergy prevention would still be active, and to which
extend, in complex matrices such as nutritional composition for infants or young children.
[0017] There is a need to bring relief in the symptoms of food allergies in populations
of infants and young children that have a history of allergic episodes and/or are
allergic.
[0018] There is a need to provide a complete nutritional composition that not only brings
a variety of nutrients but also reduces the severity of allergic reactions.
[0019] There is a need for providing a nutritional composition modulating the allergic reaction
of young patients suffering from light to moderate forms of allergies, as these patients
have special needs dictated by the low maturity of their intestinal and immune systems.
[0020] There is also a need for modulating allergic reactions in young children that do
not tolerate stringent pharmaceutical molecules, for example by nutritional interventions
such as including modulators of the immune system into the regular nutritional regimen.
[0021] There is a need for providing a nutritional composition modulating the allergic reaction
of young patients at the time, and around the time of weaning when the intestinal
tract undergoes severe modification and when new potentially allergenic solid foods
are introduced and hence the patient is particularly susceptible to the sensitization
to food allergens.
[0022] There is a need for alleviating the symptoms of allergies by providing an effective
composition, possibly by reducing the exposure to allergens, even though the composition
may not directly reduce the sensitization to allergens.
[0023] There is a need for a composition that has a positive effect on secondary prevention
of allergy while not necessarily acting on the primary prevention to the same allergies.
[0024] There is finally a need for a composition, most particularly suited for young patients
that by reduction of symptoms can help to diminish the "allergic phenotype" and thus
can lower sensitization later in life to new allergens. There is a need for attenuating
the atopic march.
Summary of the Invention
[0025] The invention is as defined by the appended claims.
[0026] In a first aspect, the present invention provides a complete nutritional composition
for reducing the symptoms of allergies originating from food in patients having allergies
triggered by food allergens, especially in infants and/or young children.
[0027] In a second aspect, the present invention provides a composition that significantly
promotes secondary prevention of allergic reactions triggered by food allergens, optionally
while not affecting the primary prevention against the same allergens.
[0028] the present invention provides a composition that is enriched in polyphenols, especially
in quercetin.
[0029] In another aspect of the invention the composition of the invention is especially
effective for infants and/or young children at the time of weaning.
[0030] The invention further extends to reduction of the sensitization to other allergens
later in life.
Brief Description of the Drawing
[0031]
Figure 1: Schematic description of OVA food allergy mouse model
Figure 2: Reduced symptoms of food allergy in mice receiving 1% of apple extract during
the last week before allergen challenge. Results of 2 independent experiments.
Figure 3: Reduced symptoms of food allergy in mice receiving 1% of apple extract (Pomactiv
HFV or Diana Naturals AFPOMM9080) either during sensitization period (primary) or
during the last week before allergen challenge (secondary).
Figure 4: Median levels of Mouse Mast-Cell Protease 1 (MMCP-1) in sera 4 hours after
challenge.
Figure 5 / Table 1: Gene expression levels in ileal Peyer's patches
Figure 6 / Table 2. Gene expression levels in ileum
Detailed Description of the Invention
[0032] Definitions: In this specification, the following terms have the following meanings:
"Complete nutritional composition ". For the purpose of this document a complete nutritional composition is a composition
that comprises a significant amount, usually 50% or more, of the major nutritional
nutrients recommended for a given age. Such major nutrients are usually provided in
quantity and proportion such as to fulfil 50% or more of the specific recommended
nutrient's doses for a given age, when used in adequate quantity for providing the
recommended caloric intake for a given age. A complete nutritional composition usually
comprises a source of protein, a source of lipid, a source of carbohydrates in a balanced
proportion that meets the general recommendation for a given age. It usually also
include micronutrients such as vitamins and minerals, as well as a source of essential
amino acids and a source of essential fatty acids. It is however understood that a
complete nutritional composition may not comprise all specific nutrients, nor all
recommended amounts, to fulfil all nutritional needs of an infant or young child.
A complete nutritional composition excludes compositions comprising merely apple extracts
or apple extracts in a predominant proportion.
"Symptoms of allergies" generally include symptoms triggered by allergens. Such symptoms include cutaneous
(redness of skin, rash, itchiness, dermatitis, eczema), ocular (itching and watering
of the eyes), gastrointestinal (congestion, abdominal pain, cramps, vomiting diarrhea),
respiratory (itching of the nose, nasal congestion, rhinitis, asthma) and in severe
cases systemic (dizziness, mental confusion, anaphylaxis) manifestations.
"Primary prevention of allergies" means all measures aiming at avoidance or reduction of allergic (immunological) sensitization
for example prevention or reduction of specific IgE antibodies.
"Secondary prevention of allergies" means prevention of the development of allergic disease/allergic symptoms in a sensitized
individual.
"Weaning period" is the period during which infants are adapting from pure liquid nutrition to solid
or semi-solid food, and adapting from quasi unique food type (generally mother milk
or infant formula) to a variety of foods.
"Sensitization " means induction/development of allergen-specific IgE antibodies.
"Children / Young children / infants / Babies" : Since children or infants are in particular affected by food allergies, the composition
of the present invention is intended for, but not limited to, children and/or infants.
According to the definitions appearing in Article 2 of the European Commission Directive
2006/141/EC of 22 December 2006 on infant formulae and follow-on formulae "Infants"
are children under the age of 12 months and young children are children between 1
and 3 years of age. The term "children" comprises the age groups from 1 to 14 years.
Babies may be equivalent to infants and/or young children.
[0033] The inventors have evidenced that allergic reaction and symptoms can be alleviated
when sensitized young mammals are provided with a complete nutritional composition
that comprises an apple extract. This defined a positive effect on the secondary prevention
of allergies.
[0034] Surprisingly this effect was not accompanied by a reduction of the sensitization
of the young mammals to allergens (i.e. no primary prevention of allergies was evidenced).
Effect of the composition:
[0035] The invention relates to the use of an apple extract comprising polyphenols for the
manufacture of a complete nutritional composition for reducing the symptoms of allergies
originating from food in patients having allergies triggered by food allergens. The
inventors have evidenced that the apple extract is capable to reduce the symptoms
of food allergy in a group of mice receiving a nutritional composition containing
apple extract. This is evidenced when an allergic reaction (challenge) is induced
after sensitization. The model mimics food allergy in humans, when humans (typically
infants/young children) are naturally sensitized to food allergens. The apple extract
hence shows a protective effect. Without being bound by the theory it is believed
that polyphenols of the apple extract interact with the complete nutritional composition
(in particular the fatty or proteinic components) to deliver an enhanced effect.
[0036] In one embodiment of the invention the effect of the composition is more Specifically
an effect on the secondary prevention of allergies. The symptoms of allergies, in
the mouse model, are indeed reduced significantly, as illustrated by a lower allergic
clinical score. Generally the symptoms can include all or a selection of the usually
recognized symptoms of allergies.
[0037] In one embodiment of the invention the symptoms comprise diarrhea, skin irritation
or respiratory symptoms or combination thereof.
[0038] In one embodiment of the invention the symptoms are accompanied by release of biochemical
mediators, such a tryptase, chymase, histamine, leukotriens.
[0039] The invention the composition however has no effect on the sensitization of the patients
to the allergens. Indeed primary prevention seems not to be achieved by the composition
comprising apple extract. The patients are still sensitized to the food allergens,
but develop less symptoms of allergy when re-exposed to the sensitizing allergen.
Without being bound to the theory it is believed that the effect therefore does not
reduce the natural immune defences (which have a positive effect for the patient)
but simply reduces the expression of the symptoms of allergy.
[0040] In one embodiment the composition reduces the sensitization to other allergens later
in life. It is believed that the composition promotes (or at least does not block)
the natural processes of immune maturation and hence, is able to have a long term
effect on lowering the sensitization to the same allergens or to different allergens
(reduction of sensitization later in life). By solely reducing the symptoms of allergy
(secondary prevention), it is hypothesized that the composition of the invention can
allow for the natural immune maturation to occur, and have such long term beneficial
effect.
Composition of the invention:
[0041] The composition comprises an apple extract that is rich in polyphenols. The apple
extract is indeed enriched in polyphenols in comparison to a crude apple extract (without
enrichment). The enrichment factor is at least 1.5, at least 2 or at least 5. It appears
critical to the effect of the composition that the apple extract is sufficiently concentrated
(enriched in polyphenols) to permit development and exertion of the anti-allergic
effect. The effect could be mediated by blocking the release of allergic mediators
from effector cells such as mast-cells (an effect comparable to anti-histaminic drugs).
Without being bound by the theory, it is also believed that some of the compounds
present in significant amount in the apple extract can bind the proteinic allergens
and mask them form being recognized by the host immune system. It can not be excluded
that the apple extract (possibly the active polyphenols) react with the specific proteins
of the composition (proteins present in the composition - especially the milk-derived
proteins) to form complexes and to mask the potential allergens.
[0042] The composition comprises between 0.1 % (w/w) and 1% (w/w) of apple extract. While
it is crucial that a sufficient amount of apple extract is present to induce the desired
effect, a quantity of apple extract that would be too high would have a negative effect
by negatively impacting the composition (un-balance of nutrients). This means, complexing
too much protein could alter the composition's content of bioavailable protein...
[0043] In one embodiment of the invention the apple extract is the protein extract commercialized
by Val de Vire Bioactives (Conde sur Vire, France) under the reference "Pomactiv HFV".
In one embodiment the apple extract is similar to or derived from the cited Pomactiv
HFV and has similar effect.
[0044] In one embodiment of the invention the composition comprises quercetin. Quercetin
may be comprised in the apple extract, in particular in the polyphenolic components
of the apple extract. Specifically quercetin can bind/complex proteins of the complete
nutritional composition including allergenic proteins contained in the composition
and the complexing can contribute to the anti-allergic effect by sequestering the
allergens from the immune system.
[0045] In one embodiment of the invention the composition comprises hydrolyzed or partially
hydrolyzed proteins. A nutritional composition based on (partially) hydrolyzed proteins
is particularly suited to the immune system and gastro-intestinal tract of infants/young
children because hydrolyzed proteins are more easily digested and have reduced allergenicity
compared to intact proteins. Furthermore it can be hypothesized that hydrolyzed proteins
can be very efficiently bound by the polyphenols of the apple extracts - for example
because of their relative size. This may trigger an enhanced masking effect of the
allergens and thus may further reduce the allergenicity of the composition. This represents
a most appropriate composition for the immune system and gastro-intestinal tract of
a food-allergic infant/young child. In such instance the combination of the apple
extract with hydrolyzed proteins is of highest relevance to the invention.
[0046] In one embodiment the hydrolyzed proteins are proteins hydrolyzed from whey proteins
and/or casein. In one embodiment the hydrolyzed proteins results from the action of
trypsin and/or chymotrypsin on the proteins (esp. whey proteins). In one embodiment
the hydrolyzed proteins comprise soy proteins or/and egg proteins. In one embodiment
the hydrolyzed proteins results from the action of proteases such as Protamex® and/or
Flavourzyme® (Novozyme, Denmark). In one embodiment the proteins of the composition,
preferably the hydrolyzed proteins, comprise cereal or eggs proteins. In one embodiment
the hydrolyzed proteins results from the action of Alcalase. The composition of the
invention can comprise a mix of 2 or more of the cited protein sources.
Target group:
[0047] The composition of the invention is most suitably targeted at relatively young patients,
although an effect on adults can not be excluded. Preferentially indeed the patients
are sufficiently young to still going through a maturation phase of their immune system
and their gastro-intestinal tract. In such patients the effect of the composition
can be more intense or more rapid. In one embodiment the composition is an infant
formula, an follow up formula, a growing-up milk, an infant cereal and/or a baby food.
Follow-up formula are usually understood as compositions for infants after the 6 first
months of life. Growing-up milk are usually understood as compositions for toddlers
and/or children between 1 or 2 and 6 years of age or between 2 or 3 and 5 years. Growing-up
milk are usually cow milk-based compositions. Preferably the composition is targeted
at infants or young children or young humans below the age of 6, between birth and
the age of 3, or between birth and weaning. In one embodiment the composition is a
starter or a follow-up infant formula. Preferably the nutritional composition comprises
a vast majority of the necessary nutrients for the feeding of the young humans.
[0048] In one embodiment the nutritional composition is an infant cereal composition for
infants/young children I to 4 years old. The composition can be more specifically
targeted to and administered during the weaning period and/or up to 12 months thereafter.
The weaning period is indeed important in regard to the invention as the infants are
exposed to a variety of foods during the weaning period, while undergoing a rapid
maturation of their immune system and their gastro-intestinal tract. Effective control
of the allergic response is therefore of particular importance during that period.
[0049] In one embodiment the nutritional composition of the invention is a growing-up milk
targeted to and specifically formulated for toddlers between 1 and 3 or children between
3 and 6 years of age. The composition is based an cow milk and may contain probiotics
and/or prebiotics. The composition can be part of a continuing regimen between 1 and
6 and be administered both during period of 1 to 3 and then between 3 and 6 years
of age. Such administration may not be daily but may be sufficiently regular (e.g.
at least once every month or week) to induce the intended effect. By acting at the
key period(s) of immune system maturation and by being administered over a long period
of time, the effect of the composition is more prominent.
[0050] In one embodiment the composition is administered both during early infancy (first
6 months) and/or 6months-1 year and during older infancy (e.g. 1 to 4 years or 2 to
6 years). Such administration can but may not be daily but may be sufficiently regular
(e.g. at least once every month or week) to induce the intended effect. A continuous
administration over a long period of time (a few years), although relatively infrequent
(one per week, once to 4 times per month), may enhance the observed effect by acting
at the critical phase of the immune system maturation.
[0051] In regard to the use of the composition of the invention the children, babies or
infants can be allergic children. Allergic children are those children, babies or
infants having experienced at least one episode of allergic reaction - light, moderate
or severe - to a food allergen. In one embodiment of the invention the children, babies
or infants have declared severe allergies to food allergens and/or have experienced
more than one moderate or severe episode of food allergy.
[0052] The food allergens encompassed by the present invention can include all types of
allergens naturally occurring or usually occurring in food, especially food for young
humans (e.g. infants, babies, children).
Experimental results:
[0053] In vitro studies with human and rodent cells have suggested that anti-oxidants may modulate
mast-cell function. As fruits and vegetables are rich in anti-oxidants we addressed
whether symptoms of food allergy could be influenced with a fruit extract. Specifically,
we tested the effect of consuming apple extract Pomactiv HFV in a mouse food allergy
model (OVA Food Allergy Mouse Model, illustrated in Fig 1).
[0054] After challenge mice treated with apple extract developed less severe clinical symptoms
than sensitized, untreated animals (Positive Control). These results are illustrated
by the experimental results shown in Figures 2 and 3:
[0055] Figure 2 shows the results of 2 independent exploratory experiments conducted with
10 animals/group. The measured output (allergic score) shows a positive trend in the
reduction of the allergy score in mice having received Apple extract in comparison
to the positive control. (p= 0.08, 0.06 respectively).
[0056] Figure 3 shows the results of a statistically powered experiment (n=15/group) in
the same model. The effect of reduction of the allergy score in the group receiving
Apple extract during the last week before challenge (secondary prevention) was confirmed
and was statistically significant (Fig 3; p=0.014, group "apple extract Pomactiv HFV,
secondary). A group treated with the same apple extract during the sensitization phase
(group "apple extract Pomactiv HFV, primary) did not show reduced symptoms after challenge.
Likewise, a group treated with a different apple extract did not have reduced symptoms
after challenge (Fig. 3, group Diana Naturals AFPOMM9080).
[0057] In addition to the clinical score, mouse mast-cell protease 1 (MMCP-1) serum levels
were determined as a measure of intestinal mast-cell activation by allergen challenge.
Consumption of apple extract Pomactiv HFV significantly reduced MMCP-1 serum levels
compared to non-treated control animals (Figure 4). Expression levels in the intestine
of various genes associated with allergy were determined by quantitative real-time
polymerase chain-reaction (qRT-PCR - see method below). The measurements of gene expression
were performed on tissues from 2 different sites (Ileal Peyer's Patch and ileum for
the positive control group, the negative control group and the group receiving Apple
extract (Pomactiv HFV) during the last week before challenge (secondary prevention).
Figure 5 (Table 1) and Figure 6 (Table 2) illustrate the results obtained: The group
receiving Apple extract Pomactiv HFV exhibited a reduced expression of various genes,
including cytokines interleukin (IL)-5, IL-13, IL-10, interferon (IFN)-γ and chemokine
CCL11 (eotaxin) in ileal Peyer's Patches and cytokines IL-13, IL-10, IL-12, IFN-γ,
chemokine CCL11 (eotaxin) and transcription factor GATA-3 in ileum. The genes for
which reduced expression has been evidenced are generally recognized markers for a
T helper 2 phenotype (IL-5, IL-13, CCL11, GATA-3) which is known to drive development
of allergies. Hence the experiments demonstrate the down-modulation of immune responses
by reduction of expression of relevant genes when the sensitized animals received
Apple extract (secondary prevention). This is believed to be contributing to the anti-allergic
effect of the tested Apple extract in the reduction of allergic symptoms (secondary
prevention).
Conclusion:
[0058] These data show that consumption of a polyphenol enriched apple extract (Pomactiv
HFV) by a sensitized animal leads to reduced allergic symptoms upon exposure to the
sensitizing allergen. The reduction of symptoms went along with reduced serum levels
of MMCP-1 and reduced expression of key genes related to allergy in the intestine,
suggesting that a modulation of components of the immune system, such as mast-cells,
contributes to the protective effect.
Analytical methods:
OVA food allergy mouse model:
[0059] Six weeks old adult conventional BALB/c mice were sensitized by the oral route (with
a gavage needle) at weekly intervals with 20 mg of Ovalbumin (OVA) from Fluka (Buchs,
Switzerland) + 10 µg/mouse of Cholera toxin (used as adjuvant) from LuBioscience (Lucerne,
Switzerland) during 7 weeks. One week after the last sensitization an oral challenge
with 100 mg of OVA was performed. Nutritional intervention with apple extract HFV
from Val de Vire Bioactives (Val de Vire Bioactives - BP2 - 50890 Conde sur Vire,
France, catalogue reference: "Pomactiv HFV") or AFPOMM9080 from Diana Naturals (Antrain,
France) (1% w/w) incorporated into the food (Kliba 3200, Kliba Nafag Kaiseraugst,
Switzerland) was done all along the experiment; as a primary prevention during sensitisation
period or as a secondary prevention starting at the end of the sensitization phase
(Fig. 1). Fifteen minutes after challenge mice were individually observed during 30
min. Clinical symptoms were recorded and quantified as follows (Allergic Score): 0:
no symptoms, less than 4 episodes of scratching; 1: 4-10 episodes of scratching around
the nose and head, no diarrhoea; 2: more than 10 episodes of scratching or bristled
fur and immobility or soft stool; 3: diarrhoea or laboured respiration or cyanosis;
4: diarrhoea in combination with immobility after prodding, bristled fur, laboured
respiration or cyanosis; 5: anaphylaxis. Four hours after challenge mice were sacrificed
(cervical dislocation), blood and the last centimetre of ileum plus one Peyer's patch
located in the same area were taken and frozen in liquid nitrogen.
Serum MMCP-1:
[0060] Murine mast cells protease 1 (MMCP-1) was quantified in mouse serum by ELISA, purchased
from Moredun Scientific (Midlothian, Scotland) according to the manufacture's instructions.
The MMCP-1 concentration was obtained by converting OD values in pg/ml using a polynomial
standard curve.
Low Density Array analyses of gene expression in intestine:
RNA extraction and quantification:
[0061] Total ribonucleique acids (RNA) from Ileum and Peyer's patch were extracted according
to the manufacture's protocol using SV Total RNA isolation System kit purchased from
Promega (Dübendorf, Switzerland).
[0062] RNA was quantified with quant-IT Ribogreen Reagent kit provided (Promega Dübendorf,
Switzerland) according to the manufacture's protocol.
Reverse Transcription:
[0063] Reverse transcription was performed on I µg of total RNA by using the Multiscribe
Reverse Transcriptase kit from Applied Biosystems (Foster City USA). Total RNA was
mixed with 50 µM of random hexamers, 0.5 mM of dNTPs, 20U of RNase inhibitor (Applied
Biosystems), 62.5U of Multiscribe reverse transcriptase, 1X RT buffer, and 5.5 mM
of MgCl2 in a final volume of 50 µl. Reverse transcription was run on a T3 thermocycler
(Biometra, Göttingen, Germany) with the following cycle program: 10 min at 25°C, 30min
at 48°C, 5 min at 95°C to finish at 4°C.
LDA:
[0064] Low Density Arrays were designed online on the Applied Biosytems website (http://www3.appliedbiosystems.com/index.htm).
The load, the run and the analysis were performed according to the manufacturer's
protocol on a quantitative ABI-Prism 7900HT.
[0065] The quantification was normalized with the mean of 3 houses keeping genes: β-actin.
GAPDH and HPRT. Based on the Cycle threshold (Ct) values obtained, a relative and
normalized mRNA expression was determined for each gene using the ΔCt. The Ct value
for each gene was corrected by the Ct mean of the three house keeping genes. The results
were calculated as a relative expression using the formula -2-ΔCt x K were K is a
10
6 factor. Fold increase results expression was normalized to expression levels in the
Negative group.
1. The use of an apple extract comprising polyphenols for the manufacture of a complete
nutritional composition for reducing the symptoms of allergies originating from food
in patients having allergies triggered by food allergens wherein said composition
has no effect on the sensitization of said patients to said allergens wherein said
apple extract is enriched in polyphenols by a factor of at least 1.5 versus a crude
apple extract wherein said composition comprises between 0.1% w/w to 1% w/w of said
apple extract.
2. The use of claim 1 wherein said composition provides significant secondary prevention
against allergic reactions triggered by said food allergens.
3. The use of any of the preceding claims wherein said composition comprises quercetin.
4. The use of any of the preceding claims wherein said composition comprises hydrolyzed
proteins.
5. The use of any of the preceding claims wherein said composition reduces the sensitization
to other allergens later in life.
6. The use of any of the preceding claims wherein said apple extract is Pomactiv HFV
from Val de Vire Bioactives (Conde sur Vire, France).
7. The use of any of the preceding claims wherein said symptoms are diarrhea, skin irritation
or respiratory or combination thereof.
8. The use of any of the preceding claims wherein said composition comprises cereal proteins.
9. The use of any of the preceding claims wherein said composition is an infant formula,
a follow-up formula, a growing-up milk, an infant cereal or a baby food.
10. The use of any of the preceding claims wherein said composition is an infant cereal
composition for infant 1 to 4 years old.
11. The use of any of the preceding claims wherein said composition is provided to infants
during the weaning period and/or up to 12 months thereafter.
12. The use of any of the preceding claims wherein said patients are infants or young
children below the age of six, preferably between birth and the age of 3.
13. The use of any of the preceding claims where said symptoms are accompanied by release
of biochemical mediators, such a tryptase, chymase, histamin, leukotriens.
1. Verwendung eines Apfelextraktes mit Polyphenolen für die Herstellung einer vollwertigen
Nahrungszusammensetzung zur Reduzierung der Allergiesymptome aus Lebensmitteln bei
Patienten mit Allergien, die durch Lebensmittelallergene ausgelöst werden, wobei die
Zusammensetzung keine Auswirkung auf die Sensibilisierung der Patienten gegenüber
Allergenen hat, wobei der Apfelextrakt mit einem Faktor von mindestens 1,5 gegenüber
einem rohen Apfelextrakt mit Polyphenolen angereichert ist, wobei die Zusammensetzung
zwischen 0,1% w/w bis 1% w/w des Apfelextraktes aufweist.
2. Verwendung nach Anspruch 1, wobei die Zusammensetzung eine wesentliche sekundäre Vorbeugung
gegen allergische Reaktionen liefert, die durch Lebensmittelallergene ausgelöst sind.
3. Verwendung nach einem der vorhergehenden Ansprüche, wobei die Zusammensetzung Quercetin
aufweist.
4. Verwendung nach einem der vorhergehenden Ansprüche, wobei die Zusammensetzung hydrolysierte
Proteine aufweist.
5. Verwendung nach einem der vorhergehenden Ansprüche, wobei die Zusammensetzung die
Sensibilisierung gegenüber anderen Allergenen im späteren Leben reduziert.
6. Verwendung nach einem der vorhergehenden Ansprüche, wobei der Apfelextrakt Pomactiv
HFV von Val de Vire Bioactives (Condé-sur-Vire, Frankreich) ist.
7. Verwendung nach einem der vorhergehenden Ansprüche, wobei die Symptome Diarrhö, Hautreizungen
oder Atemwegssymptome oder Kombinationen daraus sind.
8. Verwendung nach einem der vorhergehenden Ansprüche, wobei die Zusammensetzung Proteine
aus Getreide aufweist.
9. Verwendung nach einem der vorhergehenden Ansprüche, wobei die Zusammensetzung eine
Säuglingsnahrung, eine Folgenahrung, eine Milch für Kleinkinder, eine Getreidenahrung
für Kleinkinder oder eine Babynahrung ist.
10. Verwendung nach einem der vorhergehenden Ansprüche, wobei die Zusammensetzung eine
Getreidenahrungszusammensetzung für Kleinkinder von 1 bis 4 Jahren ist.
11. Verwendung nach einem der vorhergehenden Ansprüche, wobei die Zusammensetzung Kleinkindern
während der Entwöhnung und/oder bis zu 12 Monate danach bereitgestellt wird.
12. Verwendung nach einem der vorhergehenden Ansprüche, wobei die Patienten Säuglinge
oder Kleinkinder unter sechs Jahren, vorzugsweise zwischen der Geburt und dem Alter
von 3 Jahren sind.
13. Verwendung nach einem der vorhergehenden Ansprüche, wobei die Symptome durch die Freisetzung
von biochemischen Mediatoren, wie z.B. Tryptase, Chymase, Histamin, Leukotriene begleitet
sind.
1. Utilisation d'un extrait de pomme comprenant des polyphénols pour la fabrication d'une
composition nutritionnelle complète pour réduire les symptômes d'allergies provenant
de nourriture chez des patients ayant des allergies déclenchées par des allergènes
alimentaires, dans laquelle ladite composition n'a pas d'effet sur la sensibilisation
desdits patients auxdits allergènes,
dans laquelle ledit extrait de pomme est enrichi en polyphénols par un facteur d'au
moins 1,5 par rapport à un extrait de pomme brut,
dans laquelle ladite composition comprend entre 0,1% en poids et 1% en poids dudit
extrait de pomme.
2. Utilisation selon la revendication 1, dans laquelle ladite composition fournit une
prévention secondaire significative contre des réactions allergiques déclenchées par
lesdits allergènes alimentaires.
3. Utilisation selon l'une quelconque des revendications précédentes, dans laquelle ladite
composition comprend de la quercétine.
4. Utilisation selon l'une quelconque des revendications précédentes, dans laquelle ladite
composition comprend des protéines hydrolysées.
5. Utilisation selon l'une quelconque des revendications précédentes, dans laquelle ladite
composition réduit la sensibilisation à d'autres allergènes plus tard dans la vie.
6. Utilisation selon l'une quelconque des revendications précédentes, dans laquelle ledit
extrait de pomme est Pomactiv HFV de Val de Vire Bioactives (Condé-sur-Vire, France).
7. Utilisation selon l'une quelconque des revendications précédentes, dans laquelle lesdits
symptômes sont une diarrhée, une irritation de la peau ou des symptômes respiratoires
ou une combinaison de ceux-ci.
8. Utilisation selon l'une quelconque des revendications précédentes, dans laquelle ladite
composition comprend des protéines de céréale.
9. Utilisation selon l'une quelconque des revendications précédentes, dans laquelle ladite
composition est une préparation pour nourrisson, une préparation de suite, un lait
de croissance, de la nourriture à base de céréales ou de la nourriture pour bébé.
10. Utilisation selon l'une quelconque des revendications précédentes, dans laquelle ladite
composition est une composition à base de céréales pour nourrisson âgé de 1 à 4 ans.
11. Utilisation selon l'une quelconque des revendications précédentes, dans laquelle ladite
composition est donnée à des nourrissons au cours de la période de sevrage et/ou jusqu'à
12 mois après.
12. Utilisation selon l'une quelconque des revendications précédentes, dans laquelle lesdits
patients sont des nourrissons ou des jeunes enfants de moins de six ans, de préférence
entre la naissance et l'âge de 3 ans.
13. Utilisation selon l'une quelconque des revendications précédentes, dans laquelle lesdits
symptômes sont accompagnés par une libération de médiateurs biochimiques, comme par
exemple tryptase, chymase, histamine, leucotriènes.